Interactions of calcineurin A, calcineurin B, and Ca2+

Calcineurin B (CN-B) is the Ca2+-binding, regulatory subunit of the phosphatase calcineurin. Point mutations to Ca2+-binding sites in CN-B were generated to disable individual Ca2+-binding sites and evaluate contributions from each site to calcineurin heterodimer formation. Ca2+-binding properties of four CN-B mutants and wild-type CN-B were analyzed by flow dialysis confirming that each CN-B mutant binds three Ca2+ and that wild-type CN-B binds four Ca2+. Macroscopic dissociation constants indicate that N-terminal Ca2+-binding sites have lower affinity for Ca2+ than the C-terminal sites. Each CN-B mutant was coexpressed with the catalytic subunit of calcineurin, CN-A, to produce heterodimers with specific disruption of one Ca2+-binding site. Enzymes containing CN-B with a mutation in Ca2+-binding sites 1 or 2 have a lower ratio of CN-B to CN-A and a lower phosphatase activity than those containing wild-type CN-B or mutants in sites 3 or 4. Effects of heterodimer formation on Ca2+ binding were assessed by monitoring Ca-45(2+) exchange by flow dialysis. Enzymes containing wild-type CN-B and mutants in sites 1 and 2 exchange Ca-45(2+) slowly from two sites whereas mutants in sites 3 and 4 exchange Ca-45(2+) slowly from a single site, These data indicate that the Ca2+ bound to sites 1 and 2 is likely to vary with Ca2+ concentration and may act in dynamic modulation of enzyme function, whereas Ca2+-binding sites 3 and 4 are saturated at all times and that Ca2+ bound to these sites is structural.